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Immunological-based diagnostics provides an important tool in detecting a variety of disease conditions. This is especially the case given the specificity of components within the immune system. Notwithstanding this specificity, immunological-based diagnostics are not necessarily always sensitive enough to detect low levels of adaptive and/or innate immune activity, such as in response to a low grade infection or in the presence of a persistent low level infection or in subjects exhibiting immunodeficiency or any form of immunosuppression. There is a need to develop diagnostic assays with enhanced sensitivity in relation to cell-mediated immunoresponsiveness due to adaptive and innate immune potential. This is particularly the case in subjects exposed to or exhibiting disease conditions and agents which induce or are associated with immunosuppression.
One form of immunological-based diagnostic assays involves the stimulation of T-cells with antigens or mitogens in either isolated cell culture or whole blood culture followed by the detection of effector molecules such as cytokines produced by the activated T-cells (also referred to as effector T-cells). The effector molecules are generally detected using techniques such as enzyme immunoassays, multiplex bead analysis, ELISpot and flow cytometry. Such assays are useful for detecting disease-specific T-cell responses.
The ability to assess cell-mediated immune responsiveness of a subject is particularly important in managing immunodeficiency and immunosuppression. Immunodeficiency is characterized by a reduced ability to effectively mount an immune response This compromized or absent response can result from a primary or acquired (secondary) immunodeficiency.
Primary immunodeficiencies (PIDs) are genetically inherited and characterized by deficiencies of distinct components of the adaptive or innate immune system (Hu and Gatti, Curr Opin Allergy Clin Immunol. 8(6):540-546, 2008). Nonetheless, most immunodeficiencies are acquired (secondary) and can be induced by a pathogenic agent, as occurs with HIV infection; induced by drugs, as in immunosuppressive treatment following organ transplantation; induced by disease conditions, as can occur in cancer (e.g. leukemia, lymphoma); or induced by environmental contaminants and pollutants.
The molecular basis of immunodeficiency is diverse, however, cell-mediated immunity plays a key role in mediating many of the observed clinical manifestations. At present, immunodeficiency syndromes are diagnosed and managed in an ad hoc manner depending on the causal agent.
For example, the primary hallmark of HIV infection is progressive loss of CD4+ T-cells and impaired global immune function including an impaired antigen-specific adaptive immune response to pathogens such as cytomegalovirus (CMV) and Hepatitis B virus (HBV) [Douek et al, Annu. Rev. Med. 60:471-84, 2009 and Chang et al, J. Virol. 83:7649-58, 2009]. Hence, CD4+ T-cell count and viral load remain the key surrogate markers of HIV infection and have been extensively validated as predictive of both immune dysfunction and with progressive infection and immune reconstitution following prophylaxis (Hengel and Kovacs, J. Infect. Dis. 188(12):1791-3, 2003). However, many instances exist in which clinical disease does not correlate with the level of immune function as predicted by these surrogate markers particularly when opportunistic infections develop in the presence of normal CD4+ T-cell counts and low viral loads (Solomon et al, J. Infect. Dis. 187:1915-23, 2003).
Similarly, monitoring the cellular immunodeficiency status of patients that have undergone solid organ transplants (SOTs) and are receiving medications to suppress their immune system (immunosuppressants) as an anti-rejection measure, are also managed in an ad hoc manner. Typically, patient immunosuppressant drug levels are monitored as are regular blood counts and incidence of graft, nosocomial or community-acquired infections (Schrem et al, Dtsch Arztebl Int. 106(9):148-156, 2009).
The inherent problem associated with many of the current markers used to monitor and define immunodeficiency associated with disease states is the absence of cell-mediated immunity. A number of T-cell function tests have been developed that measure lymphoproliferative responses to mitogens such as phytohemagglutinin (PHA), pokeweed mitogen and concanavalinA (ConA). However, these only measure the functional ability of T-cells; a subset of cells involved in cell-mediated immunity. Importantly, it has become increasingly evident that innate immune mechanisms contribute greatly to host defence, either through acting alone or by enhancing specific T-cell responses (Cooper et al, Hematology:314-30, 2003). Therefore, the functional responses of innate (natural killer [NK] cell) and adaptive (T-cell) immune cells together form a more comprehensive analysis of cell-mediated immunity.
The ability to assess cell-mediated immunity is of clinical importance. For example, in excess of 33 million adults and children are infected with HIV (2008 Report on the Global AIDS Epidemic, UNAIDS; ISBN 978 92 9 173711 6) and currently around 100,000 solid organ transplantations are performed per year worldwide (Matesanz et al, Transplant Proc. 41(6):2297-301, 2009).
Innate immune dysregulation has been implicated in HIV infection with a whole blood IFN-γ assay described for the monitoring of innate immune responsiveness to the TLR-7/8 agonist compound (a imidazoquinoline compound, 8848) [Nowroozalizadeh et al, Cytokine 46:325-31, 2009]. Furthermore, a diagnostic device that measures functional T-cell responses (Cylex ImmuKnow (Registered Trade Mark), as described in US 2003/0199006 A 1) measures ATP production from PHA-stimulated CD4+cells (Kowalski et al, J Immunotoxicol. 4(3):225-32, 2007). Specifically, the Cylex assay has demonstrated clinical utility in the SOT setting such that adverse events including infection and rejection can be adequately managed.
There is a need to develop an assay which can assess the combined functional status of innate and adaptive cell-mediated immunoresponsiveness in a subject and to determine disease conditions and agents which induce immunosuppression.